CN111394477B - Reagent kit for detecting 120 gene loci based on second-generation sequencing technology and primer combination used by reagent kit - Google Patents

Reagent kit for detecting 120 gene loci based on second-generation sequencing technology and primer combination used by reagent kit Download PDF

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CN111394477B
CN111394477B CN202010249140.8A CN202010249140A CN111394477B CN 111394477 B CN111394477 B CN 111394477B CN 202010249140 A CN202010249140 A CN 202010249140A CN 111394477 B CN111394477 B CN 111394477B
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王乐
郭立亮
康克莱
张驰
季安全
叶健
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Institute of Forensic Science Ministry of Public Security PRC
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Abstract

The invention discloses a reagent kit for detecting 120 loci based on a second-generation sequencing technology and a primer combination used by the reagent kit. The primer combination comprises 230 primers, wherein the nucleotide sequences of the 230 primers are shown as SEQ ID NO: 1-SEQ ID NO: 230, respectively. The length of the amplicon in 120 loci detected by the kit provided by the invention is only 2.5% (3/120) which is larger than 300bp, thus being beneficial to the analysis of degradation test materials. Experiments prove that the kit provided by the invention is adopted to detect STR typing of 120 loci in 2800M standard, and the typing result is accurate. The invention has important application value.

Description

Reagent kit for detecting 120 gene loci based on second-generation sequencing technology and primer combination used by reagent kit
Technical Field
The invention belongs to the technical field of forensic medicine, and particularly relates to a kit for detecting 120 loci based on a second-generation sequencing technology and a primer combination used by the kit, in particular to a kit for detecting 119 STR loci and Amelogenin loci based on a second-generation sequencing technology and a primer combination used by the kit.
Background
STR typing technology is simple and rapid, results are reliable, sensitivity is high, repeatability is good, and the STR typing technology is a main tool for forensic genetics research. The STR typing mainly adopts the technical means of carrying out genotyping through capillary electrophoresis detection, and the technology realizes the judgment and the reading of the genotype through detecting the total fluorescence intensity of DNA molecules of the same genotype, on one hand, the technology can only carry out qualitative analysis on the genotype and can not accurately quantify the proportion of the genotype, and on the other hand, the technology can limit the number of loci which can be detected by STR typing. The second-generation sequencing STR typing technology has larger information content and higher data accuracy, not only can obtain the length information of STR, but also can obtain complete sequence information, finds out alleles with inconsistent sequences in more fragments with the same length, and can also obtain all locus information to be detected at one time, thereby avoiding multiple capillary electrophoresis typing misoperation, ensuring that the obtained typing result comes from the same sample, shortening the time, saving the cost and simultaneously improving the detection accuracy.
Human Y chromosome short tandem repeats (Y-STR) refer to short tandem repeats of non-recombinant regions present on the Y chromosome. The Y-STR has three characteristics of male specificity, paternal inheritance, haplotype inheritance and the like, and is widely used for male family investigation, individual identification, tracing origin, migration, fusion and the like of a paternal population in population genetics research. Human X-chromosome short tandem repeats (X-STR) refer to short tandem repeats of non-recombinant regions present on the X-chromosome. The X-STR genetic characteristics are different from autosomes and Y chromosomes, show as sex linkage characteristics, have unique value in the case of identifying the special genetic relationship such as father-mother relationship, mother-son relationship, alternate genetic relationship, random relationship and the like, and can be used as an important supplement for the autosomes STR and Y-STR. Therefore, the autosomal STR, the Y-STR and the X-STR are subjected to combined typing, so that the information content of DNA detection in forensic science can be remarkably improved.
At present, two kits which are most widely applied to second-generation sequencing STR typing products and contain the STR typing sites are respectively products of Illumina company and Thermo Fisher company. First, both are very expensive; secondly, the available information has limitations, only the data and analysis of the inherent loci in the products can be obtained, and the number of loci is less, especially the number of X-STR loci is less; thirdly, due to the species difference, the products are not completely suitable for STR population genetic polymorphism analysis of the population in China. A huge DNA database is established in China, so that the forensic medicine second-generation sequencing STR typing reagent for screening loci based on population genetics data of people in China, researching and developing more loci in combined autosome, Y chromosome and X chromosome has very important research significance and application value.
Disclosure of Invention
The invention aims to prepare a kit suitable for STR population genetic polymorphism analysis of Chinese population.
The invention firstly protects a primer combination, which can comprise a primer 1-a primer 230; primer 1-primer 230 are single-stranded DNA molecules, and the nucleotide sequences can be shown as SEQ ID NO: 1-SEQ ID NO: 230, respectively.
The primer combination can specifically consist of a primer 1-a primer 230.
In any of the above primer combinations, the molar ratio of primer 1 to primer 230 may be specifically 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.04: 0.04: 0.04: 0.04: 0.12: 0.12: 0.04: 0.04: 0.08: 0.08: 0.16: 0.16: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.06: 0.06: 0.06: 0.06: 0.08: 0.08: 0.08: 0.08: 0.16: 0.16: 0.24: 0.24: 0.48: 0.48: 0.24: 0.24: 0.04: 0.04: 0.08: 0.08: 0.12: 0.12: 0.48: 0.48: 0.12: 0.12: 0.16: 0.16: 0.08: 0.08: 0.12: 0.12: 0.06: 0.06: 0.12: 0.12: 0.12: 0.12: 0.06: 0.06: 0.16: 0.16: 0.08: 0.08: 0.12: 0.12: 0.12: 0.12: 0.24: 0.24: 0.04: 0.04: 0.08: 0.08: 0.02: 0.02: 0.02: 0.02: 0.08: 0.08: 0.04: 0.04: 0.16: 0.16: 0.02: 0.02: 0.08: 0.08: 0.08: 0.08: 0.03: 0.03: 0.03: 0.03: 0.08: 0.08: 0.04: 0.04: 0.16: 0.16: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.02: 0.02: 0.08: 0.08: 0.04: 0.04: 0.04: 0.04: 0.08: 0.08: 0.08: 0.08: 0.04: 0.04: 0.08: 0.08: 0.02: 0.02: 0.08: 0.08: 0.02: 0.02: 0.04: 0.04: 0.04: 0.04: 0.02: 0.02: 0.08: 0.08: 0.12: 0.12: 0.02: 0.02: 0.08: 0.08: 0.16: 0.16: 0.04: 0.04: 0.24: 0.24: 0.08: 0.08: 0.24: 0.24: 0.05: 0.05: 0.08: 0.08: 0.12: 0.12: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.16: 0.16: 0.08: 0.08: 0.08: 0.08: 0.16: 0.16: 0.12: 0.12: 0.08: 0.08: 0.24: 0.24: 0.12: 0.12: 0.04: 0.04: 0.16: 0.16: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.12: 0.12: 0.24: 0.24: 0.12: 0.12: 0.12: 0.12: 0.08: 0.08: 0.04: 0.04: 0.08: 0.08: 0.32: 0.32: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08.
any of the primer combinations described above can be used to amplify 120 loci.
The invention also discloses a composite amplification system based on 120 loci, which can comprise any one of the primer combinations.
The composite amplification system can be composed of any one of the primer combinations.
The concentrations of the primers 1 to 230 in the multiplex amplification system may be 0.08. mu.M, 0.04. mu.M, 0.12. mu.M, 0.04. mu.M, 0.08. mu.M, 0.16. mu.M, 0.08. mu.M, 0.06. mu.06. mu.M, 0.06. mu.08, 0.08. mu.M, 0.24. mu.M, 0.08, 0.M, 0.48. mu.M, 0.48. mu.08, 0.M, 0.08, 0.M, 0.48. mu.M, 0.M, 0.24. mu.48, 0.08, 0.M, 0.48. mu.M, 0.48, 0.M, 0.48. mu.M, 0.M, 0.48, 0.M, 0.48. mu.08. mu.M, 0.48, 0., 0.12. mu.M, 0.06. mu.M, 0.12. mu.M, 0.06. mu.M, 0.16. mu.M, 0.08. mu.M, 0.12. mu.M, 0.24. mu.M, 0.04. mu.M, 0.08. mu.M, 0.02. mu.M, 0.08. mu.M, 0.04. mu.M, 0.16. mu.M, 0.02. mu.02. mu.08, 0.08. mu.08, 0.M, 0.04. mu.M, 0.16. mu.08, 0.M, 0.04, 0.08. mu.08, 0.08, 0.04, 0.08. mu.08, 0.08, 0.M, 0.04, 0.08. mu.08, 0.M, 0.08. mu.M, 0.08. mu.08, 0.04, 0.M, 0.04, 0.08, 0.04, 0.08, 0.04, 0.M, 0.04, 0.08, 0.04, 0.M, 0.04, 0.08, 0.04, 0.08, 0.M, 0.08, 0.M, 0.08, 0.04, 0.08, 0.04, 0.M, 0.08, 0.04, 0.08, 0.04, 0.08, 0.08. mu.M, 0.04. mu.M, 0.08. mu.M, 0.02. mu.M, 0.08. mu.M, 0.02. mu.M, 0.04. mu.M, 0.02. mu.M, 0.08. mu.M, 0.12. mu.M, 0.02. mu.M, 0.08. mu.M, 0.16. mu.M, 0.04. mu.M, 0.24. mu.M, 0.08. mu.08. mu.M, 0.24. mu.M, 0.08. mu.08, 0.08. mu.M, 0.08. mu.M, 0.16. mu.08, 0.M, 0.08. mu.M, 0.08. mu.08, 0.M, 0.24. mu.08, 0.M, 0.08. mu.M, 0.08. mu.08, 0.M, 0.08. mu.M, 0.08 μ M, 0.08. mu.M, 0.24. mu.M, 0.08, 0.M, 0.08. mu.M, 0.24. mu.08, 0.M, 0.08. mu.M, 0.08, 0.M, 0.08, 0.0.0.M, 0.M, 0.08, 0.M, 0.24. mu.M, 0.0.08, 0.M, 0.08, 0.M, 0.08. mu.08, 0.08. mu.M, 0.M, 0.08, 0.M, 0.08. mu.M, 0.08, 0.M, 0.24. mu.M, 0.M, 0.24. mu.08. mu.M, 0.08, 0.M, 0.08. mu.M, 0.08 μ M, 0.0.0.M, 0.0.0.0.0.M, 0.24. mu.0.0.M, 0.M, 0.0.0.08. mu.M, 0.08, 0.M, 0.0.24, 0.0.08. mu.0.0.0.M, 0.0.M, 0.0.0.0.08, 0.M, 0.08, 0.M, 0.0, 0.24. mu.M, 0.12. mu.M, 0.04. mu.M, 0.16. mu.M, 0.08. mu.M, 0.12. mu.M, 0.24. mu.M, 0.12. mu.M, 0.08. mu.M, 0.04. mu.M, 0.08. mu.M, 0.32. mu.M, 0.08. mu.08, 0.08. mu.M, 0.08. mu.08, 0.M, 0.08. mu.08, and 0.08. mu.08.
The composite amplification system can also comprise reagents required for carrying out PCR amplification reaction; the "reagents required for performing a PCR amplification reaction" does not include primers required for a PCR amplification reaction.
Any one of the composite amplification systems can be 20 mu L and consists of 10 mu L of PCR Master Mix, any one of the primer combinations and the template. The PCR Master Mix can be specifically a product of the national institute of public Security department material evidence authentication center. The template may be specifically a1 ng/. mu.L aqueous solution of 2800M standard or genomic DNA of a sample. The sample may be a human saliva collection card. The standard 2800M may be a product of Promega corporation, catalog number DD 7101.
A kit containing any primer combination is also in the protection scope of the invention; the use of the kit can be (h1) or (h2) or (h3) or (h 4): (h1) STR parting; (h2) typing an autosomal STR; (h3) Y-STR typing; (h4) and (4) carrying out X-STR typing.
The invention also provides a preparation method of any one of the above composite amplification systems or any one of the above kits; the preparation method comprises the step of packaging each primer in any one primer combination individually.
The invention also protects the application of any one of the primer combinations or any one of the composite amplification systems in the preparation of the kit; the use of the kit can be (h1) or (h2) or (h3) or (h 4): (h1) STR parting; (h2) typing an autosomal STR; (h3) Y-STR typing; (h4) and (4) carrying out X-STR typing.
The invention also protects the application of any one of the primer combinations or any one of the multiplex amplification systems, which can be (h1) or (h2) or (h3) or (h 4): (h1) STR parting; (h2) typing an autosomal STR; (h3) Y-STR typing; (h4) and (4) carrying out X-STR typing.
Any of the above-described 120 loci may be selected from Amelogenin, CSF1PO, FGA, TH01, TPOX, VWA, Penta D, Penta E, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, DYS19, DYS385 2/B, DYS389-I, DYS-II, DYS390, DYS391, DYS392, DYS393, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS576, DYS635, Y-GATA-H4, D1S 7, D1S1677, D2S 1677, DYS 124456, DYS 2S 1082D 1082S 1083D 1082S 1082D 10853, DYS 1082S 1083D 1082S 1083D 1082S 1083D 1082D 1083D 1082S 1083D 1083, DXS 1082S 1083D 1082S 1083, DX S1082D 1083D 1082D 1083, DXS 1083D 1082D 1083, DX 3D 1083D 1082S 1082D 1083, DXS 1082S 1083, DX 3, DXS 1082S 1082D 1082S 1082D 1083, DXS 1082D 1082S 1082D 1083, DXS 1082S 1083, DX 3D 1082S 1082D 1082S 1083, DX 3D 1082S 1082D 1082S 1083, DXS 1082S 1083, DX 3, DX 3, DXS 1082D 1083, DXS 1082D 1082S 1082D 1082S 1083, DXS 1082D 1083D 1082S 1082D 1083, DXS 1082D 1082S 1082D 1083, DXS 1082S 1083, DXS 1083, DX 3D 1083, DXS 1082S 1082D 1082S 1082D 1082S 1082D 1082S 1083, DXS 1082S 1082D 1082S 1083, DX, DXS 1083, D1082S 1083, DXS 1082D 1083, DXS 1082S 1083, DXS 1082D 1082S 1083, DXS 1082D 1083, D1082S 1083, D1082D 1083, DXS 1082D 1083, D1082D 1083, DXS 1083, D1082S 1082D 108, DXS7132, DXS7423, DXS7424, DXS981, DXS9895, DXS9902, GATA165B12, GATA31E08 and HPRTB. Wherein CSF1PO, FGA, TH01, TPOX, VWA, Penta D, Penta E, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D1S1627, D1S1677, D2S1360, D2S1776, D2S441, D3S1744, D3S3045, D3S3053, D3S4529, D4S2364, D4S2366, D5S2500, D5S2800, D6S474, D6S 1122, D7S3048, D8S1132, D9S 215482, D9S 2364, D10S1248, D10S 1245S 23210S 2325, D6S474, D6S 2322S 10819, D18S 10819S 10820, D18S 10819, D3627, D18S 10819, D10819S 10814, D3627, D11S 10814, D11S 10819S 10814 and D11S 10819S 10814S 10820. DYS19, DYS385a/b, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS576, DYS635, Y-GATA-H4, DYS388, DYS399S1, DYS404S1a/b, DYS504, DYS505, DYS508, DYS520, DYS531, DYS549, DYS552, DYS641, DYS570, DYS593, DYS596, DYS622, DYS643 and DYS STR are 41Y-loci. DXS10079, DXS10103, DXS6789, DXS6799, DXS6800, DXS6801, DXS6804, DXS6809, DXS7130, DXS7132, DXS7423, DXS7424, DXS981, DXS9895, DXS9902, GATA165B12, GATA31E08 and HPRTB are 18X-STR loci. Amelogenin is a sex-determining locus. DYS385a/b and DYS404S1a/b respectively comprise two typing fragments. DYS399S1 contained three typing fragments.
The kit for detecting 120 loci based on the second-generation sequencing technology comprises 60 autosomal STR loci, 41Y-STR loci, 18X-STR loci and 1 individuation determination locus Amelogenin, and can detect more genetic information. In addition, the length of the amplicon in 120 loci in the composite amplification system provided by the invention is only 2.5 percent (3/120) which is larger than 300bp, while the length of the amplicon in the ForenSeq system is only 2.5 percentTMThe amplicon length in the DNA Signature Prep Kit is greater than about 20.3% of 300bp (12/59), which is not conducive to analysis of degraded samples. Experiments prove that the kit provided by the invention is adopted to detect STR typing of 120 loci in 2800M standard, and the typing result is accurate. The invention has important application value.
Drawings
FIG. 1 shows the length range distribution of PCR amplification products for 119 STR loci.
FIG. 2 is a species-specific assay of the kit prepared in example 1.
FIG. 3 is a graph showing the sensitivity detection of the kit prepared in example 1.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
The PCR Master Mix is a product of the material evidence authentication center of the Ministry of public Security. Standard 2800M is a product from Promega corporation under the catalog number DD 7101. The Agencourt AMPure XP 5mL Kit is a product of Beckman Coulter company, and the product number is A63880. QubitTMThe dsDNA HS Assay Kit is a product of Thermo Fisher, Inc., having a product number of Q32854. NanoDrop 2000 UV Spectrophotometer is a product of Thermo Fisher corporation. TruSeq DNA PCR-Free HT Library Prep Kit is a product of Illumina, Inc., and the product number is FC-121-3003. KAPA Library Quantification Kit is a product of KAPA corporation, cat # KK 4824. Miseq reagent kit v3-600 cycles sequencing reagent is a product of Illumina, Inc., with the cargo number MS-102-3003. The Miseq FGx second generation sequencer is a product of Illumina corporation.
Example 1 preparation of a kit for detecting 120 loci (including 60 autosomal STR loci, 41Y-STR loci, 18X-STR loci, and 1 individualized Amelogenin locus) based on a second-generation sequencing technique
Screening of one, 120 loci
The inventor of the invention obtains a large number of candidate autosomal STR loci, Y-STR loci and X-STR loci by consulting documents and the existing second-generation sequencing STR typing products, and finally obtains 120 loci including 60 autosomal STR loci, 41Y-STR loci, 18X-STR loci and 1 individuality determining locus Amelogenin by screening according to the STR population genetic polymorphism analysis result of Chinese population.
The 60 autosomal STR loci are: CSF1PO, FGA, TH01, TPOX, VWA, Penta D, Penta E, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D1S1627, D1S1677, D2S1360, D2S1776, D2S441, D3S1744, D3S3045, D3S3053, D3S4529, D4S2364, D4S2366, D5S2500, D5S2800, D6S474, D6S477, D7S3048, D8S1132, D9S2157, D9S 477, D10S1248, D10S 1245, D14310S 1435, D23210S 2325S 1080, D6S474, D6S477, D7S3048, D8S1132, D215482, D9S2157, D9S 1088, D10S1248, D S10819S 973S 10819, D6520, D3S 973S 10819, D3S 10819, D10819S 10819, D3S 10819, D3S 1083S 10819, D1088, D3S 1083S 1089S 1083S 1088, D10819, D1088, D1089S 1083S 10819, D1089S 1083S 1089S 1088, D1083S 1088, D3S 1088, D1089S 1088, D10819, D1083S 1089S 1083S 1089S 1083, D1089S 1083S 1089S 1088, D3S 1089S 1083S 1088, D3S 1083, D3S 1089S 1088, D1083S 1089S 1083S 1088, D1083S 1083, D1088, D10810S 1088, D10810S 1083S 10810S 1089S 1083S 10810S 1088, D10810.
The 41Y-STR loci are respectively: DYS19, DYS385a/b, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS576, DYS635, Y-GATA-H4, DYS388, DYS399S1, DYS404S1a/b, DYS504, DYS505, DYS508, DYS520, DYS531, DYS549, DYS552, DYS641, DYS570, DYS593, DYS596, DYS622, DYS643, DYS 645; wherein, DYS385a/b and DYS404S1a/b respectively comprise two typing fragments, and DYS399S1 comprises three typing fragments.
The 18X-STR loci are respectively: DXS10079, DXS10103, DXS6789, DXS6799, DXS6800, DXS6801, DXS6804, DXS6809, DXS7130, DXS7132, DXS7423, DXS7424, DXS981, DXS9895, DXS9902, GATA165B12, GATA31E08, HPRTB.
Preparation of primer combination
1. Primers for amplifying each locus (preferably, amplification length is 300bp or less) are artificially designed and synthesized, and then PCR amplification is performed to obtain a specific amplification product for each locus.
2. And (3) combining the amplification conditions of the single gene locus, selecting a proper amplification program, and carrying out composite amplification. Because the number of complex loci is large and the mutual inhibition condition among primers is complex, the loci need to be eliminated one by one, found out, redesigned and synthesized. In addition, primer dimers are formed between primers at different loci, which reduces the amplification efficiency of the primers and requires redesign and synthesis of primers.
3. During primary composite amplification, the concentration of each primer in a reaction system is 0.1 mu M; and then adjusting the concentration of the primers to obtain the optimal concentration of each primer during PCR amplification.
The primer combination is obtained through the steps. The primer combination consists of 230 primers and is used for detecting 120 loci. The names of the individual loci, the names of the primers corresponding to the amplification loci, the nucleotide sequences of the primers, and the allelic typing ranges are shown in Table 1 in sequence in columns 1, 2, 3, and 6. The optimal concentration of each primer when performing PCR amplification is shown in column 5 of Table 1.
TABLE 1
Figure BDA0002434844870000051
Figure BDA0002434844870000061
Figure BDA0002434844870000071
Figure BDA0002434844870000081
Figure BDA0002434844870000091
Figure BDA0002434844870000101
The length and nucleotide sequence of PCR amplification products for each locus in the hg38 human reference genome (for information on the hg38 human genome, see the website http:// hgdownloadload. soe. ucsc. edu/goldenPath/hg38/bigZips/hg38.2bit) are detailed in Table 2 (exemplary). The distribution of the length ranges of the PCR amplification products for each STR locus is shown in FIG. 1. It can be seen that the amplicon length greater than 300bp in 120 loci is only 2.5% (3/120), whereas ForenceSeqTMThe amplicon length in the DNA Signature Prep Kit is greater than about 20.3% of 300bp (12/59).
TABLE 2
Figure BDA0002434844870000111
Figure BDA0002434844870000121
Figure BDA0002434844870000131
Figure BDA0002434844870000141
Figure BDA0002434844870000151
Figure BDA0002434844870000161
Figure BDA0002434844870000171
Figure BDA0002434844870000181
Figure BDA0002434844870000191
Figure BDA0002434844870000201
Preparation of reagent kit for detecting 120 gene loci based on second-generation sequencing technology
The kit for detecting 120 loci based on the second generation sequencing technology comprises a primer mixture; the primer mixture is formed by mixing 230 primers prepared in the step two.
Example 2 STR typing of 2800M Standard test substance Using the kit prepared in example 1
1. DNA sample preparation
A2800M aqueous solution of the standard 2800M was diluted with ultrapure water to give a1 ng/. mu.L aqueous solution of the standard 2800M.
2. PCR amplification
And (3) performing PCR amplification by using a 2800M aqueous solution serving as a template and the primer mixture prepared in the third step of the example 1 to obtain a PCR amplification product.
The reaction system was 20. mu.L, consisting of 10. mu.L of PCR Master Mix, 1. mu.L of 2800M aqueous standard, primer Mix and ribozyme-free water. The concentration of each primer in the reaction system is shown in column 5 of Table 1.
Reaction procedure: 5min at 95 ℃; 30s at 98 ℃, 90s at 60 ℃, 1min at 72 ℃ and 28 cycles; 10min at 72 ℃; storing at 4 ℃.
3. Purification and quantification
(1) And (3) taking the PCR amplification product, and purifying according to the instruction steps of an Agencourt AMPure XP 5mL Kit to obtain a PCR purified product.
(2) The PCR purified product was subjected to the following QubitTMThe dsDNA HS Assay Kit instructions, using a NanoDrop 2000 UV spectrophotometer for quantification, to obtain the concentration of PCR purified product.
4. Library preparation
Taking the PCR purified product, sequentially carrying out terminal repair, terminal repair product purification, connection A-tail, connection Adapter and connection product purification according to the operation steps of the instruction manual of the TruSeq DNA PCR-Free HT Library Prep Kit, then carrying out Library Quantification and Library standardization according to the instruction manual of the KAPA Library Quantification Kit, and finishing Library preparation.
5. On-machine testing
Taking the library prepared in the step 4, sequencing the library on a Miseq FGx second-generation sequencer by using a Miseq reagent kit v3-600 cycles sequencing reagent, and performing data analysis after the sequencing is finished.
The results are shown in Table 3. The result shows that the standard 2800M obtains complete STR typing and can completely meet the requirements of forensic STR inspection.
TABLE 3
Figure BDA0002434844870000211
Figure BDA0002434844870000221
Figure BDA0002434844870000231
Figure BDA0002434844870000241
Note: n and the following numbers indicate a base sequence, and the following numbers indicate the number of bases in the sequence.
Example 3, accuracy verification of the kit prepared in example 1
1. Taking 1ng standard 2800M, and respectively making into desired dosage forms according to Identifier Plus kit (Thermo Fisher) and Global FilerTMA kit (Thermo Fisher),
Figure BDA0002434844870000243
Yfiler Plus kit (Thermo Fisher), PowerPlex21 kit (Promega), and DNATypeTMCapillary electrophoresis detection was performed in the protocol of the Y26 kit (center for Material evidence identification by the department of public Security) to obtain the allelic genotypes at each locus. The results of the typing are shown in Table 4, column 2.
2. Standard 2800M was assayed according to the procedure of example 2 to obtain the allelic genotype of the locus. The results of the typing are shown in Table 4, column 3.
The results show that the loci of the kit prepared in example 1, which coincide with the capillary electrophoresis typing kit described above, are completely identical in typing results in the 2800M standard sample (column 4 of Table 4).
TABLE 4
Figure BDA0002434844870000242
Figure BDA0002434844870000251
Figure BDA0002434844870000261
Note: "-" means absent.
Example 4 species-specific detection of the kits prepared in example 1
1. The 2800M aqueous solution of the standard sample of example 2 was replaced with E.coli, fish, chicken, duck, cat, dog, cow, sheep, pig or monkey genomic DNA, all without further modification. The concentrations of the genomic DNA of Escherichia coli, the genomic DNA of fish, the genomic DNA of chicken, the genomic DNA of duck, the genomic DNA of cat, the genomic DNA of dog, the genomic DNA of cow, the genomic DNA of sheep, the genomic DNA of pig and the genomic DNA of monkey were all 1 ng/. mu.L.
2. Standard 2800M was assayed according to the procedure of example 2.
3. And drawing a map by using the categories of the autosomal STR locus, the Y-STR locus and the X-STR locus as abscissa and the length type of the allele as ordinate.
The results are shown in FIG. 2(aSTRs are autosomal STR loci). The results show that the kit prepared in example 1 has good species specificity.
Example 5, sensitivity detection of the kit prepared in example 1
1. A2800M sample was diluted with ultrapure water to obtain a 2800M aqueous solution 1 at a concentration of 5 ng/. mu.L, a 2800M aqueous solution 2at a concentration of 2 ng/. mu.L, a 2800M aqueous solution 3 at a concentration of 1 ng/. mu.L, a 2800M aqueous solution 4 at a concentration of 500 pg/. mu.L, a 2800M aqueous solution 5 at a concentration of 200 pg/. mu.L, a 2800M aqueous solution 6 at a concentration of 100 pg/. mu.L, and a 2800M aqueous solution 7 at a concentration of 50 pg/. mu.L, respectively.
2. The standard 2800M aqueous solution in example 2 was replaced with standard 2800M aqueous solution 1, standard 2800M aqueous solution 2, standard 2800M aqueous solution 3, standard 2800M aqueous solution 4, standard 2800M aqueous solution 5, standard 2800M aqueous solution 6, or standard 2800M aqueous solution 7, and the other steps were not changed.
3. And (3) respectively counting the proportions of the alleles in the autosomal STR locus, the Y-STR locus and the X-STR locus according to the sequencing result in the step (2) (aiming at reflecting the integrity of the typing result).
The results are shown in FIG. 3(aSTRs are autosomal STR loci). The result shows that the sensitivity of the kit prepared in the embodiment 1 is higher, and the detection rates of the alleles of the autosomal STR, the Y-STR and the X-STR are all 100% when the DNA input amount is 500 pg/reaction system or more; when the input amount of DNA is 200 pg/reaction system, the detection rate of autosomal STR alleles is more than 98%, and the detection rate of Y-STR alleles and X-STR alleles reaches 100%.
Example 6 application of the kit prepared in example 1-typing of sample loci with saliva Collection cards
The first sample, the second sample, the third sample, the fourth sample and the fifth sample are genome DNAs of different human saliva collecting cards, and the concentrations of the genome DNAs are all 1 ng/mu L. All saliva acquisition cards are provided by the construction of a daily DNA database of a material evidence identification center of the ministry of public Security.
The 2800M aqueous solution standard of example 2 was replaced with sample one, sample two, sample three, sample four, or sample five, all other steps remaining unchanged.
The results of the test of sample one are shown in Table 5. The results of the second sample are shown in Table 6. The results of the measurement of sample three are shown in Table 7. The results of the test of sample four are shown in Table 8. The results of the test of sample five are shown in Table 9. The results show that the complete STR typing results are obtained for sample one, sample two, sample three, sample four and sample five.
TABLE 5
Figure BDA0002434844870000271
Figure BDA0002434844870000281
Figure BDA0002434844870000291
Figure BDA0002434844870000301
Note: n and the following numbers indicate a base sequence, and the following numbers indicate the number of bases in the sequence.
TABLE 6
Figure BDA0002434844870000302
Figure BDA0002434844870000311
Figure BDA0002434844870000321
Figure BDA0002434844870000331
Note: n and the following numbers indicate a base sequence, and the following numbers indicate the number of bases in the sequence.
TABLE 7
Figure BDA0002434844870000332
Figure BDA0002434844870000341
Figure BDA0002434844870000351
Figure BDA0002434844870000361
Note: n and the following numbers indicate a base sequence, and the following numbers indicate the number of bases in the sequence.
TABLE 8
Figure BDA0002434844870000362
Figure BDA0002434844870000371
Figure BDA0002434844870000381
Figure BDA0002434844870000391
Note: n and the following numbers indicate a base sequence, and the following numbers indicate the number of bases in the sequence.
TABLE 9
Figure BDA0002434844870000392
Figure BDA0002434844870000401
Figure BDA0002434844870000411
Figure BDA0002434844870000421
Note: n and the following numbers indicate a base sequence, and the following numbers indicate the number of bases in the sequence.
Example 7, the kit prepared in example 1 has irreplaceability of the primer
The kit prepared in example 1 is obtained by the present inventors through a large number of experiments, and mainly includes repeated debugging of primers for amplifying each locus and investigation of primer concentration. Because the number of the compound loci is large and the mutual inhibition among the primers is complex, the primers for amplifying each locus in the kit cannot be replaced.
One, increasing loci leads to suppression of loci in the original system
The number of loci in the system is gradually increased in the process of preparing the kit, and the situation that the existing loci or newly-increased loci in the system are inhibited can be met when the loci are added to the established system every time. Taking the case of increasing the number of loci at one time, the details are as follows:
1. the established system comprises 91 loci (90 STR loci and Amelogenin loci), and primers for amplifying 37 loci thereof are shown in table 10, and the remaining 54 loci are CSF1PO, TH01, TPOX, VWA, Penta D, D3S1358, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS576, D1S1677, D2S1776, D2S441, D3S3053, D6S474, D10S1435, D11S4463, D20S482, D22S1045, DYS505, DYS508, DYS520, DYS596, DYS643, DYS 7114303, DXS 14331, DXS 31, DXS 121, DXS 31, DXS 6831, DXS 31, DXS 6831, DXS 26S 2S 102, DXS 42, DXS 120S 6831, DXS 42, DXS 26S 2S 645, DXS 42, DXS 26, DXS 26, DXS 26, DXS 26, DXS9 DXS 26, DXS 26 DXS9 DXS 26, DXS 26, DXS9 DXS 26, DXS 26, DXS9 DXS 26, DXS9 DXS 26, DXS. 27 STR loci were newly added, and primers 497-658 for amplifying the 27 STR loci shown in Table 11 were designed and synthesized. Primer 497-primer 550 make up primer set 1, primer 551-primer 604 make up primer set 2, and primer 605-primer 658 make up primer set 3.
Watch 10
Figure BDA0002434844870000422
Figure BDA0002434844870000431
Figure BDA0002434844870000441
TABLE 11
Figure BDA0002434844870000442
Figure BDA0002434844870000451
Figure BDA0002434844870000461
Figure BDA0002434844870000471
2. The primer combinations shown in table 10 were combined with primer combination 1, primer combination 2, and primer combination 3, respectively, to obtain three primer mixtures.
3. A2800M aqueous solution of the standard 2800M was diluted with ultrapure water to give a1 ng/. mu.L aqueous solution of the standard 2800M.
4. And (3) performing PCR amplification by respectively adopting the three primer mixtures in the step (2) by taking a standard 2800M aqueous solution as a template to obtain PCR amplification products. The concentration of each primer in the reaction system was 0.1. mu.M.
5. The same procedure as in step 3 of example 2.
6. Same as step 4 of example 2.
7. Same as step 5 in example 2.
The results are shown in Table 12. The results show that many loci in the original system influence each other with the newly increased loci, the existing loci are inhibited differently under the influence of the primer combination 1, the primer combination 2 and the primer combination 3, and the three groups of primers of the newly increased loci are different in performance and inhibited.
TABLE 12
Figure BDA0002434844870000472
Figure BDA0002434844870000481
Figure BDA0002434844870000491
Note: x indicates no allele was obtained, -indicates absence.
Secondly, randomly replacing primers of the kit loci to cause the loci in the system to be inhibited
Random replacement of the primers at the kit loci can lead to inhibition of some loci in the system, including loci with and without primer replacement.
1. Primers 659-704 for amplification of the loci shown in Table 13 were artificially designed and synthesized. Primer 659-primer 684 make up primer set a, and primer 685-primer 704 make up primer set b.
Watch 13
Figure BDA0002434844870000492
Figure BDA0002434844870000501
2. The primers of the loci involved in the primer combination a were substituted for the primers of the corresponding loci in the primer combination of example 1 to obtain a primer combination A. And (b) replacing the primers of the corresponding loci in the primer combination in the example 1 by the primers of the loci related to the primer combination b to obtain a primer combination B.
The primer combination A and the primer combination B are both composed of 230 primers.
3. A2800M aqueous solution of the standard 2800M was diluted with ultrapure water to give a1 ng/. mu.L aqueous solution of the standard 2800M.
4. And (3) performing PCR amplification by using a 2800M aqueous solution of a standard substance as a template and adopting a primer mixture A or a primer mixture B to obtain a PCR amplification product. The primer mixture A is formed by mixing 230 primers in the primer combination A. The primer mixture B is formed by mixing 230 primers in the primer combination B.
The reaction system was 20. mu.L, consisting of 10. mu.L of PCR Master Mix, 1. mu.L of 2800M aqueous standard, ribozyme-free water, a mixture (primer mixture A or primer mixture B), and. In this reaction system, the concentrations of primers at loci related to both primer set a and primer set b were 0.1. mu.M, and the concentrations of the other primers are shown in column 5 of Table 1.
Reaction procedure: 5min at 95 ℃; 30s at 98 ℃, 90s at 60 ℃, 1min at 72 ℃ and 28 cycles; 10min at 72 ℃; storing at 4 ℃.
5. The same procedure as in step 3 of example 2.
6. Same as step 4 of example 2.
7. Same as step 5 in example 2.
The results are shown in Table 14. The results show that random replacement of primers in the kit prepared in example 1 results in inhibition of some loci, and the inhibition is different.
TABLE 14
Figure BDA0002434844870000502
Figure BDA0002434844870000511
Figure BDA0002434844870000521
Note: and x represents no allele obtained.
The above results indicate that the primers in the kit prepared in example 1 are not replaceable, and the inventors of the present invention obtained the primer combination and the primer concentration in the kit through a large number of experiments.
<110> material evidence identification center of public security department
<120> reagent kit for detecting 120 loci based on second generation sequencing technology and primer combination used by same
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<213>Artificial sequence
<400>86
aacatcacaa aaatcttcac 20
<210>87
<211>21
<212>DNA
<213>Artificial sequence
<400>87
ctgagccact gcctctaaaa t 21
<210>88
<211>25
<212>DNA
<213>Artificial sequence
<400>88
acttccagtc ctcacttatc tttaa 25
<210>89
<211>21
<212>DNA
<213>Artificial sequence
<400>89
aactacagaa gcagagagaa a 21
<210>90
<211>21
<212>DNA
<213>Artificial sequence
<400>90
cagttatgag gacggttgac a 21
<210>91
<211>21
<212>DNA
<213>Artificial sequence
<400>91
tctaatgagt gggtttgtga t 21
<210>92
<211>21
<212>DNA
<213>Artificial sequence
<400>92
tctttgctct catgaataga t 21
<210>93
<211>19
<212>DNA
<213>Artificial sequence
<400>93
gataaaggaa ctgttaggg 19
<210>94
<211>22
<212>DNA
<213>Artificial sequence
<400>94
ccccaaaatt acttgagcca at 22
<210>95
<211>22
<212>DNA
<213>Artificial sequence
<400>95
aacaaaacta ggagatcatg tg 22
<210>96
<211>20
<212>DNA
<213>Artificial sequence
<400>96
acaaccttga ctttgatgtg 20
<210>97
<211>22
<212>DNA
<213>Artificial sequence
<400>97
attcctaggg tgaacttcac at 22
<210>98
<211>19
<212>DNA
<213>Artificial sequence
<400>98
ataggtgcaa aactcaaag 19
<210>99
<211>19
<212>DNA
<213>Artificial sequence
<400>99
atgccactgc actccagcc 19
<210>100
<211>19
<212>DNA
<213>Artificial sequence
<400>100
cccccatctt attcactct 19
<210>101
<211>21
<212>DNA
<213>Artificial sequence
<400>101
acattatcac caatttttct a 21
<210>102
<211>17
<212>DNA
<213>Artificial sequence
<400>102
gaaaatatct cgtgggc 17
<210>103
<211>21
<212>DNA
<213>Artificial sequence
<400>103
tagtcagggt tttccaagag a 21
<210>104
<211>21
<212>DNA
<213>Artificial sequence
<400>104
agccaattcc tctcataaat c 21
<210>105
<211>19
<212>DNA
<213>Artificial sequence
<400>105
ttctgtcaca gggctgatg 19
<210>106
<211>20
<212>DNA
<213>Artificial sequence
<400>106
cctgggggat atctcaaaca 20
<210>107
<211>22
<212>DNA
<213>Artificial sequence
<400>107
agcttcacaa tgaaactcat tt 22
<210>108
<211>19
<212>DNA
<213>Artificial sequence
<400>108
cacttggaac aagtcagca 19
<210>109
<211>21
<212>DNA
<213>Artificial sequence
<400>109
ggttagtggc ttaatgtatt t 21
<210>110
<211>22
<212>DNA
<213>Artificial sequence
<400>110
tttctacttg cacaaaaaat ta 22
<210>111
<211>18
<212>DNA
<213>Artificial sequence
<400>111
cctgggtggc aaagcgag 18
<210>112
<211>20
<212>DNA
<213>Artificial sequence
<400>112
cagcaaagcc accgtgtagt 20
<210>113
<211>22
<212>DNA
<213>Artificial sequence
<400>113
tattcacata tctcattgta tt 22
<210>114
<211>18
<212>DNA
<213>Artificial sequence
<400>114
caccgagcac ccaaagac 18
<210>115
<211>19
<212>DNA
<213>Artificial sequence
<400>115
ctgagcatta gccccagga 19
<210>116
<211>19
<212>DNA
<213>Artificial sequence
<400>116
acctctgtat cccacccct 19
<210>117
<211>21
<212>DNA
<213>Artificial sequence
<400>117
acttgtgtgg tgctgtgttt g 21
<210>118
<211>21
<212>DNA
<213>Artificial sequence
<400>118
gcactccacc ttgggtcata g 21
<210>119
<211>19
<212>DNA
<213>Artificial sequence
<400>119
agctgagaga tcacgccac 19
<210>120
<211>21
<212>DNA
<213>Artificial sequence
<400>120
gcagcatgaa gctcacgaaa g 21
<210>121
<211>22
<212>DNA
<213>Artificial sequence
<400>121
ttcttatgaa atctctgtgt ct 22
<210>122
<211>22
<212>DNA
<213>Artificial sequence
<400>122
ctgcaaagga ggaacttagc ct 22
<210>123
<211>22
<212>DNA
<213>Artificial sequence
<400>123
gggaggaaaa gacataggat ag 22
<210>124
<211>22
<212>DNA
<213>Artificial sequence
<400>124
cagcctggat aacagagcga ga 22
<210>125
<211>23
<212>DNA
<213>Artificial sequence
<400>125
ccagaatcct tccagctaat aac 23
<210>126
<211>21
<212>DNA
<213>Artificial sequence
<400>126
tctttaagtg tctagagagg a 21
<210>127
<211>21
<212>DNA
<213>Artificial sequence
<400>127
caattccttg taataactct a 21
<210>128
<211>22
<212>DNA
<213>Artificial sequence
<400>128
aaaaaataac tgtagaacca at 22
<210>129
<211>21
<212>DNA
<213>Artificial sequence
<400>129
ttaattaaat tgtcaaatct g 21
<210>130
<211>22
<212>DNA
<213>Artificial sequence
<400>130
cctaaaggtt tatccatgct gt 22
<210>131
<211>21
<212>DNA
<213>Artificial sequence
<400>131
cccaacataa catattgctt a 21
<210>132
<211>23
<212>DNA
<213>Artificial sequence
<400>132
tatgtgtgga tagacacatg aca 23
<210>133
<211>18
<212>DNA
<213>Artificial sequence
<400>133
gagcctgggt gacagagc 18
<210>134
<211>23
<212>DNA
<213>Artificial sequence
<400>134
atgtgtataa caaaattcct atg 23
<210>135
<211>19
<212>DNA
<213>Artificial sequence
<400>135
agcctgggtg agagtgaga 19
<210>136
<211>21
<212>DNA
<213>Artificial sequence
<400>136
tggtggcacc caaaactgaa t 21
<210>137
<211>18
<212>DNA
<213>Artificial sequence
<400>137
gcttacacac agggcccc 18
<210>138
<211>22
<212>DNA
<213>Artificial sequence
<400>138
ctgtagaaag caacaaccct aa 22
<210>139
<211>21
<212>DNA
<213>Artificial sequence
<400>139
cttcatgtga caaaagccac a 21
<210>140
<211>22
<212>DNA
<213>Artificial sequence
<400>140
tggcaggcta agagttaccc at 22
<210>141
<211>21
<212>DNA
<213>Artificial sequence
<400>141
ttttaatact caaccaaaac t 21
<210>142
<211>21
<212>DNA
<213>Artificial sequence
<400>142
tgacgagtta atgggtgcaa c 21
<210>143
<211>19
<212>DNA
<213>Artificial sequence
<400>143
acctgctgtg gctcctcct 19
<210>144
<211>19
<212>DNA
<213>Artificial sequence
<400>144
gccctcttct gtctctcca 19
<210>145
<211>20
<212>DNA
<213>Artificial sequence
<400>145
tttactagag gtggcagaag 20
<210>146
<211>20
<212>DNA
<213>Artificial sequence
<400>146
atacctaatg cgtgcgggcc 20
<210>147
<211>21
<212>DNA
<213>Artificial sequence
<400>147
gagtgacaga gtgataccat g 21
<210>148
<211>20
<212>DNA
<213>Artificial sequence
<400>148
ggggatattt gggactcctt 20
<210>149
<211>20
<212>DNA
<213>Artificial sequence
<400>149
ggcagtgggg ctgcaaagga 20
<210>150
<211>20
<212>DNA
<213>Artificial sequence
<400>150
tgtatcagag gacagcctcc 20
<210>151
<211>21
<212>DNA
<213>Artificial sequence
<400>151
agggctacat agagaaacag a 21
<210>152
<211>19
<212>DNA
<213>Artificial sequence
<400>152
ttgtgtgagc caatccttc 19
<210>153
<211>21
<212>DNA
<213>Artificial sequence
<400>153
ccagagaaac agaaccaata g 21
<210>154
<211>21
<212>DNA
<213>Artificial sequence
<400>154
tcttgaactc atcctccata a 21
<210>155
<211>19
<212>DNA
<213>Artificial sequence
<400>155
agattttccc cgatgatag 19
<210>156
<211>20
<212>DNA
<213>Artificial sequence
<400>156
gcgaatgtat gattggcaat 20
<210>157
<211>23
<212>DNA
<213>Artificial sequence
<400>157
tcacctaata aatcccttct cat 23
<210>158
<211>19
<212>DNA
<213>Artificial sequence
<400>158
tccaatttgc atcccaacc 19
<210>159
<211>17
<212>DNA
<213>Artificial sequence
<400>159
tgagccagat cgcacca 17
<210>160
<211>20
<212>DNA
<213>Artificial sequence
<400>160
ttcatgtgag ttagccgttt 20
<210>161
<211>18
<212>DNA
<213>Artificial sequence
<400>161
cagtttgcat aggtagag 18
<210>162
<211>21
<212>DNA
<213>Artificial sequence
<400>162
aactggacag cttaggattg g 21
<210>163
<211>22
<212>DNA
<213>Artificial sequence
<400>163
tctttgacaa aaaaatgaga ca 22
<210>164
<211>20
<212>DNA
<213>Artificial sequence
<400>164
aagggcttaa gaaatttcaa 20
<210>165
<211>18
<212>DNA
<213>Artificial sequence
<400>165
agcctgggca acagagca 18
<210>166
<211>22
<212>DNA
<213>Artificial sequence
<400>166
accactgtgc caagctatta cc 22
<210>167
<211>20
<212>DNA
<213>Artificial sequence
<400>167
ttctggcgaa gtaacccaaa 20
<210>168
<211>23
<212>DNA
<213>Artificial sequence
<400>168
aaagtaagta aaggttcgag tca 23
<210>169
<211>22
<212>DNA
<213>Artificial sequence
<400>169
aatcccaaat tccagcttat ta 22
<210>170
<211>23
<212>DNA
<213>Artificial sequence
<400>170
tagattgata gaacaaataa ggt 23
<210>171
<211>19
<212>DNA
<213>Artificial sequence
<400>171
agagaacagc ctgcccaac 19
<210>172
<211>19
<212>DNA
<213>Artificial sequence
<400>172
tttcctcaac ctcccatgt 19
<210>173
<211>19
<212>DNA
<213>Artificial sequence
<400>173
ccactggcat tcaaatcct 19
<210>174
<211>19
<212>DNA
<213>Artificial sequence
<400>174
cagtgtctct tcctgggtt 19
<210>175
<211>23
<212>DNA
<213>Artificial sequence
<400>175
gcaattaggt aggtaaagag gaa 23
<210>176
<211>22
<212>DNA
<213>Artificial sequence
<400>176
atgtcccctt ttccatttgt ga 22
<210>177
<211>21
<212>DNA
<213>Artificial sequence
<400>177
gtccatagtg ccgaggtcaa g 21
<210>178
<211>22
<212>DNA
<213>Artificial sequence
<400>178
gcaaacgact gccatagata at 22
<210>179
<211>21
<212>DNA
<213>Artificial sequence
<400>179
gactattttt tctgtgccaa g 21
<210>180
<211>19
<212>DNA
<213>Artificial sequence
<400>180
accttgaggg atgccaaag 19
<210>181
<211>21
<212>DNA
<213>Artificial sequence
<400>181
ggtagaaatc ctggctgtgt c 21
<210>182
<211>19
<212>DNA
<213>Artificial sequence
<400>182
gaagggcttc taagggatg 19
<210>183
<211>20
<212>DNA
<213>Artificial sequence
<400>183
agatctcacc agtggactcc 20
<210>184
<211>21
<212>DNA
<213>Artificial sequence
<400>184
actgctgatt ggaaccactt g 21
<210>185
<211>20
<212>DNA
<213>Artificial sequence
<400>185
tgtacaggtc caaaggcagc 20
<210>186
<211>23
<212>DNA
<213>Artificial sequence
<400>186
gtctaggtat gagatgaaat tga 23
<210>187
<211>20
<212>DNA
<213>Artificial sequence
<400>187
gcactccagc ctcggtgata 20
<210>188
<211>22
<212>DNA
<213>Artificial sequence
<400>188
aatgtaggta tgaaatccag ca 22
<210>189
<211>20
<212>DNA
<213>Artificial sequence
<400>189
gcctggtgat agagagagac 20
<210>190
<211>22
<212>DNA
<213>Artificial sequence
<400>190
acctaacaat taacaatatc ac 22
<210>191
<211>20
<212>DNA
<213>Artificial sequence
<400>191
tcattgaacc tcatgctctg 20
<210>192
<211>23
<212>DNA
<213>Artificial sequence
<400>192
tctcagaaat caccactaaa gaa 23
<210>193
<211>21
<212>DNA
<213>Artificial sequence
<400>193
tggttacggg tggcaatcat a 21
<210>194
<211>17
<212>DNA
<213>Artificial sequence
<400>194
atcttggccc agcctcc 17
<210>195
<211>18
<212>DNA
<213>Artificial sequence
<400>195
ctgggtgacc aagtgaga 18
<210>196
<211>20
<212>DNA
<213>Artificial sequence
<400>196
ttttgcatca ataatcatca 20
<210>197
<211>21
<212>DNA
<213>Artificial sequence
<400>197
atagatcatg ggacttctca g 21
<210>198
<211>19
<212>DNA
<213>Artificial sequence
<400>198
gctctccagg gaaacagaa 19
<210>199
<211>20
<212>DNA
<213>Artificial sequence
<400>199
gatccctaga gggacagaac 20
<210>200
<211>20
<212>DNA
<213>Artificial sequence
<400>200
gacctcgtga tcatgtaagt 20
<210>201
<211>22
<212>DNA
<213>Artificial sequence
<400>201
ttctgagata actacttgca ca 22
<210>202
<211>20
<212>DNA
<213>Artificial sequence
<400>202
taaagacaca aaaatgcaca 20
<210>203
<211>18
<212>DNA
<213>Artificial sequence
<400>203
gggggctggt tcccctga 18
<210>204
<211>18
<212>DNA
<213>Artificial sequence
<400>204
cctattgtgg gaccttgt 18
<210>205
<211>24
<212>DNA
<213>Artificial sequence
<400>205
aactactcct ggtatcaaaa tctg 24
<210>206
<211>22
<212>DNA
<213>Artificial sequence
<400>206
cctgtaccat aatcacatga gt 22
<210>207
<211>20
<212>DNA
<213>Artificial sequence
<400>207
ccgcacctgg ccctattgaa 20
<210>208
<211>21
<212>DNA
<213>Artificial sequence
<400>208
acgctaccta cacactgtgc t 21
<210>209
<211>21
<212>DNA
<213>Artificial sequence
<400>209
ctcaaggagg accatgtttc a 21
<210>210
<211>21
<212>DNA
<213>Artificial sequence
<400>210
tcctagctca ggaatactga g 21
<210>211
<211>18
<212>DNA
<213>Artificial sequence
<400>211
gtgagccctg atcatgcc 18
<210>212
<211>20
<212>DNA
<213>Artificial sequence
<400>212
cctcccacat cagccttcct 20
<210>213
<211>19
<212>DNA
<213>Artificial sequence
<400>213
gaaccactcc tggtgccaa 19
<210>214
<211>22
<212>DNA
<213>Artificial sequence
<400>214
tccttaatag tgtgagccca tt 22
<210>215
<211>22
<212>DNA
<213>Artificial sequence
<400>215
tgtcacacaa ataaatgaat ga 22
<210>216
<211>21
<212>DNA
<213>Artificial sequence
<400>216
tgccctttat cacccagatt t 21
<210>217
<211>21
<212>DNA
<213>Artificial sequence
<400>217
gagaacatat acatttcagc a 21
<210>218
<211>18
<212>DNA
<213>Artificial sequence
<400>218
caaggcctgc ctgggaaa 18
<210>219
<211>21
<212>DNA
<213>Artificial sequence
<400>219
caccaccata ttgttccttg a 21
<210>220
<211>21
<212>DNA
<213>Artificial sequence
<400>220
gtagacatac ttctcgtttc c 21
<210>221
<211>20
<212>DNA
<213>Artificial sequence
<400>221
caattcaaga tgagatttgg 20
<210>222
<211>22
<212>DNA
<213>Artificial sequence
<400>222
tgttaggcct tttgacttgt ct 22
<210>223
<211>20
<212>DNA
<213>Artificial sequence
<400>223
gttcttgtaa cttttctgtc 20
<210>224
<211>22
<212>DNA
<213>Artificial sequence
<400>224
atacacattc atatcaggag ta 22
<210>225
<211>22
<212>DNA
<213>Artificial sequence
<400>225
atgtatcatc aatcatctat cc 22
<210>226
<211>22
<212>DNA
<213>Artificial sequence
<400>226
ctgtgtatgc tttaaaagtt ga 22
<210>227
<211>21
<212>DNA
<213>Artificial sequence
<400>227
gatgtataga cagagctggt g 21
<210>228
<211>22
<212>DNA
<213>Artificial sequence
<400>228
ggaccaagca aatcaataga aa 22
<210>229
<211>22
<212>DNA
<213>Artificial sequence
<400>229
tttccatctc tgtctccatc tt 22
<210>230
<211>21
<212>DNA
<213>Artificial sequence
<400>230
attcaataaa taggagaagg g 21

Claims (19)

1. A primer combination consisting of primer 1-primer 230; the primer 1-the primer 230 are single-stranded DNA molecules, and the nucleotide sequences are shown as SEQ ID NO: 1-SEQ ID NO: 230, respectively.
2. The primer combination of claim 1, wherein: the molar ratio of primer 1 to primer 230 was 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.04: 0.04: 0.04: 0.04: 0.12: 0.12: 0.04: 0.04: 0.08: 0.08: 0.16: 0.16: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.06: 0.06: 0.06: 0.06: 0.08: 0.08: 0.08: 0.08: 0.16: 0.16: 0.24: 0.24: 0.48: 0.48: 0.24: 0.24: 0.04: 0.04: 0.08: 0.08: 0.12: 0.12: 0.48: 0.48: 0.12: 0.12: 0.16: 0.16: 0.08: 0.08: 0.12: 0.12: 0.06: 0.06: 0.12: 0.12: 0.12: 0.12: 0.06: 0.06: 0.16: 0.16: 0.08: 0.08: 0.12: 0.12: 0.12: 0.12: 0.24: 0.24: 0.04: 0.04: 0.08: 0.08: 0.02: 0.02: 0.02: 0.02: 0.08: 0.08: 0.04: 0.04: 0.16: 0.16: 0.02: 0.02: 0.08: 0.08: 0.08: 0.08: 0.03: 0.03: 0.03: 0.03: 0.08: 0.08: 0.04: 0.04: 0.16: 0.16: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.02: 0.02: 0.08: 0.08: 0.04: 0.04: 0.04: 0.04: 0.08: 0.08: 0.08: 0.08: 0.04: 0.04: 0.08: 0.08: 0.02: 0.02: 0.08: 0.08: 0.02: 0.02: 0.04: 0.04: 0.04: 0.04: 0.02: 0.02: 0.08: 0.08: 0.12: 0.12: 0.02: 0.02: 0.08: 0.08: 0.16: 0.16: 0.04: 0.04: 0.24: 0.24: 0.08: 0.08: 0.24: 0.24: 0.05: 0.05: 0.08: 0.08: 0.12: 0.12: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.16: 0.16: 0.08: 0.08: 0.08: 0.08: 0.16: 0.16: 0.12: 0.12: 0.08: 0.08: 0.24: 0.24: 0.12: 0.12: 0.04: 0.04: 0.16: 0.16: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.12: 0.12: 0.24: 0.24: 0.12: 0.12: 0.12: 0.12: 0.08: 0.08: 0.04: 0.04: 0.08: 0.08: 0.32: 0.32: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08: 0.08.
3. a multiplex amplification system based on 120 loci consisting of the primer combination of claim 1 or 2; the 120 loci are composed of Amelogenin, CSF1PO, FGA, TH01, TPOX, VWA, Penta D, Penta E, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, DYS19, DYS385a/B, DYS 389-23 389-II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS 1082S 1082D 1082S 1082D 1082S 1082D 1083D 1082S 10853, DX D1082S 1082D 1082S 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D 1083D 1082D, DXS9895, DXS9902, GATA165B12, GATA31E08 and HPRTB.
4. The multiplex amplification system of claim 3, wherein: the concentrations of the primers 1 to 230 in the multiplex amplification system are 0.08. mu.M, 0.04. mu.M, 0.12. mu.M, 0.04. mu.M, 0.08. mu.M, 0.16. mu.M, 0.08. mu.M, 0.06. mu.06. M, 0.06. mu.08, 0.08. mu.M, 0.24. mu.M, 0.48. mu.M, 0.08, 0.M, 0.48. mu.M, 0.08, 0.48. mu.M, 0.M, 0.08, 0.M, 0.48. mu.M, 0.48, 0.M, 0.48. mu.M, 0.48, 0.M, 0., 0.12. mu.M, 0.06. mu.M, 0.12. mu.M, 0.06. mu.M, 0.16. mu.M, 0.08. mu.M, 0.12. mu.M, 0.24. mu.M, 0.04. mu.M, 0.08. mu.M, 0.02. mu.M, 0.08. mu.M, 0.04. mu.M, 0.16. mu.M, 0.02. mu.02. mu.08, 0.08. mu.08, 0.M, 0.04. mu.M, 0.16. mu.08, 0.M, 0.04, 0.08. mu.08, 0.08, 0.04, 0.08. mu.08, 0.08, 0.M, 0.04, 0.08. mu.08, 0.M, 0.08. mu.M, 0.08. mu.08, 0.04, 0.M, 0.04, 0.08, 0.04, 0.08, 0.04, 0.M, 0.04, 0.08, 0.04, 0.M, 0.04, 0.08, 0.04, 0.08, 0.M, 0.08, 0.M, 0.08, 0.04, 0.08, 0.04, 0.M, 0.08, 0.04, 0.08, 0.04, 0.08, 0.08. mu.M, 0.04. mu.M, 0.08. mu.M, 0.02. mu.M, 0.08. mu.M, 0.02. mu.M, 0.04. mu.M, 0.02. mu.M, 0.08. mu.M, 0.12. mu.M, 0.02. mu.M, 0.08. mu.M, 0.16. mu.M, 0.04. mu.M, 0.24. mu.M, 0.08. mu.08. mu.M, 0.24. mu.M, 0.08. mu.08, 0.08. mu.M, 0.08. mu.M, 0.16. mu.08, 0.M, 0.08. mu.M, 0.08. mu.08, 0.M, 0.24. mu.08, 0.M, 0.08. mu.M, 0.08. mu.08, 0.M, 0.08. mu.M, 0.08 μ M, 0.08. mu.M, 0.24. mu.M, 0.08, 0.M, 0.08. mu.M, 0.24. mu.08, 0.M, 0.08. mu.M, 0.08, 0.M, 0.08, 0.0.0.M, 0.M, 0.08, 0.M, 0.24. mu.M, 0.0.08, 0.M, 0.08, 0.M, 0.08. mu.08, 0.08. mu.M, 0.M, 0.08, 0.M, 0.08. mu.M, 0.08, 0.M, 0.24. mu.M, 0.M, 0.24. mu.08. mu.M, 0.08, 0.M, 0.08. mu.M, 0.08 μ M, 0.0.0.M, 0.0.0.0.0.M, 0.24. mu.0.0.M, 0.M, 0.0.0.08. mu.M, 0.08, 0.M, 0.0.24, 0.0.08. mu.0.0.0.M, 0.0.M, 0.0.0.0.08, 0.M, 0.08, 0.M, 0.0, 0.24. mu.M, 0.12. mu.M, 0.04. mu.M, 0.16. mu.M, 0.08. mu.M, 0.12. mu.M, 0.24. mu.M, 0.12. mu.M, 0.08. mu.M, 0.04. mu.M, 0.08. mu.M, 0.32. mu.M, 0.08. mu.08, 0.08. mu.M, 0.08. mu.08, 0.M, 0.08. mu.08, and 0.08. mu.08.
5. A multiplex amplification system based on 120 loci as defined in claim 3, consisting of the primer combination of claim 1 or 2 and reagents required for performing a PCR amplification reaction; the reagents required to perform the PCR amplification reaction do not include primers required for the PCR amplification reaction.
6. The multiplex amplification system of claim 5, wherein: the concentrations of the primers 1 to 230 in the multiplex amplification system are 0.08. mu.M, 0.04. mu.M, 0.12. mu.M, 0.04. mu.M, 0.08. mu.M, 0.16. mu.M, 0.08. mu.M, 0.06. mu.06. M, 0.06. mu.08, 0.08. mu.M, 0.24. mu.M, 0.48. mu.M, 0.08, 0.M, 0.48. mu.M, 0.08, 0.48. mu.M, 0.M, 0.08, 0.M, 0.48. mu.M, 0.48, 0.M, 0.48. mu.M, 0.48, 0.M, 0., 0.12. mu.M, 0.06. mu.M, 0.12. mu.M, 0.06. mu.M, 0.16. mu.M, 0.08. mu.M, 0.12. mu.M, 0.24. mu.M, 0.04. mu.M, 0.08. mu.M, 0.02. mu.M, 0.08. mu.M, 0.04. mu.M, 0.16. mu.M, 0.02. mu.02. mu.08, 0.08. mu.08, 0.M, 0.04. mu.M, 0.16. mu.08, 0.M, 0.04, 0.08. mu.08, 0.08, 0.04, 0.08. mu.08, 0.08, 0.M, 0.04, 0.08. mu.08, 0.M, 0.08. mu.M, 0.08. mu.08, 0.04, 0.M, 0.04, 0.08, 0.04, 0.08, 0.04, 0.M, 0.04, 0.08, 0.04, 0.M, 0.04, 0.08, 0.04, 0.08, 0.M, 0.08, 0.M, 0.08, 0.04, 0.08, 0.04, 0.M, 0.08, 0.04, 0.08, 0.04, 0.08, 0.08. mu.M, 0.04. mu.M, 0.08. mu.M, 0.02. mu.M, 0.08. mu.M, 0.02. mu.M, 0.04. mu.M, 0.02. mu.M, 0.08. mu.M, 0.12. mu.M, 0.02. mu.M, 0.08. mu.M, 0.16. mu.M, 0.04. mu.M, 0.24. mu.M, 0.08. mu.08. mu.M, 0.24. mu.M, 0.08. mu.08, 0.08. mu.M, 0.08. mu.M, 0.16. mu.08, 0.M, 0.08. mu.M, 0.08. mu.08, 0.M, 0.24. mu.08, 0.M, 0.08. mu.M, 0.08. mu.08, 0.M, 0.08. mu.M, 0.08 μ M, 0.08. mu.M, 0.24. mu.M, 0.08, 0.M, 0.08. mu.M, 0.24. mu.08, 0.M, 0.08. mu.M, 0.08, 0.M, 0.08, 0.0.0.M, 0.M, 0.08, 0.M, 0.24. mu.M, 0.0.08, 0.M, 0.08, 0.M, 0.08. mu.08, 0.08. mu.M, 0.M, 0.08, 0.M, 0.08. mu.M, 0.08, 0.M, 0.24. mu.M, 0.M, 0.24. mu.08. mu.M, 0.08, 0.M, 0.08. mu.M, 0.08 μ M, 0.0.0.M, 0.0.0.0.0.M, 0.24. mu.0.0.M, 0.M, 0.0.0.08. mu.M, 0.08, 0.M, 0.0.24, 0.0.08. mu.0.0.0.M, 0.0.M, 0.0.0.0.08, 0.M, 0.08, 0.M, 0.0, 0.24. mu.M, 0.12. mu.M, 0.04. mu.M, 0.16. mu.M, 0.08. mu.M, 0.12. mu.M, 0.24. mu.M, 0.12. mu.M, 0.08. mu.M, 0.04. mu.M, 0.08. mu.M, 0.32. mu.M, 0.08. mu.08, 0.08. mu.M, 0.08. mu.08, 0.M, 0.08. mu.08, and 0.08. mu.08.
7. A kit comprising the primer combination of claim 1 or 2; the kit is used for STR typing.
8. A kit comprising the primer combination of claim 1 or 2; the kit is used for autosomal STR typing.
9. A kit comprising the primer combination of claim 1 or 2; the kit is used for Y-STR typing.
10. A kit comprising the primer combination of claim 1 or 2; the application of the kit is X-STR typing.
11. A method for preparing a multiplex amplification system according to any one of claims 3 to 6 or a kit according to any one of claims 7 to 10, comprising the step of packaging each primer of the primer combination according to claim 1 or 2 separately.
12. Use of a primer combination according to claim 1 or 2 or a multiplex amplification system according to any one of claims 3 to 6 in the preparation of a kit; the kit is used for STR typing.
13. Use of a primer combination according to claim 1 or 2 or a multiplex amplification system according to any one of claims 3 to 6 in the preparation of a kit; the kit is used for autosomal STR typing.
14. Use of a primer combination according to claim 1 or 2 or a multiplex amplification system according to any one of claims 3 to 6 in the preparation of a kit; the kit is used for Y-STR typing.
15. Use of a primer combination according to claim 1 or 2 or a multiplex amplification system according to any one of claims 3 to 6 in the preparation of a kit; the application of the kit is X-STR typing.
16. Use of a primer combination according to claim 1 or 2 or a multiplex amplification system according to any one of claims 3 to 6 for STR typing.
17. Use of a primer combination according to claim 1 or 2 or a multiplex amplification system according to any one of claims 3 to 6 for autosomal STR typing.
18. Use of a primer combination according to claim 1 or 2 or a multiplex amplification system according to any one of claims 3 to 6 for Y-STR typing.
19. Use of a primer combination according to claim 1 or 2 or a multiplex amplification system according to any one of claims 3 to 6 for X-STR typing.
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